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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.06. - 14.07.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test substance is skin irritant, due to the test Skin Sensitisation in Guinea Pigs was performed.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: BFA
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Luboš Sobota, 298 03 Městec Králové, monitored breeding farm, RČH CZ 21760039- Age at receiving: 6-7 weeks - Weight at study initiation: 354.93 ± 32.82- Housing: sterilized shavings of soft wood, monitored conditions, microbiologically defined background, according to internal SOP No.40- Diet: pelleted standard diet for guinea pigs ad libitum (Altromin 3023 for Giunea pig)- Water: drinking tap water ad libitum (supplemented by ascorbic acid)- Acclimation period: 5 days- Number/Sex: 33 animals Pilot experiment: 3 animalsExposed group: 20 animalsControl group: 10 animals (13 males + 17 females)ENVIRONMENTAL CONDITIONSMicroclimatic conditions: - Room temperature 22 ± 3°C, permanently monitored- Relative humidity 30 – 70 %, permanently monitored- Light: 12 hour light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: 2-Mercaptobenzothiazole
Concentration / amount:
0.1 ml
Day(s)/duration:
0 - 6
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
5% test substance in vaseline
Day(s)/duration:
20
No. of animals per dose:
33 animals:Pilot experiment: 3 animalsExposed group: 20 animalsControl group: 10 animals
Details on study design:
MAIN STUDYDose levelsThe appropriate suspensions of the test substance determined from pilot experiment were as follows:Induction-intradermal injections:5% suspension of the test substance in physiological salineInduction-topical application:50% test substance in vaselineChallenge-topical application:5% test substance in vaselineA. INDUCTION EXPOSUREInduction: Intradermal InjectionsDay 0 – treated groupThree pairs of intradermal injections of 0.1 mL volume were given in the shoulder region, which was cleared of hair so that one of each pair lies on each side of the midline.Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological salineInjection 2: 5% suspension of the test substance in physiological salineInjection 3: 5% suspension of the test substance in a 1:1 mixture (v/v) FCA/physiological salineDay 0 – control groupThree pairs of intradermal injections of 0.1 mL volume were given in the same sites as in the treated animals.Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological salineInjection 2: physiological salineInjection 3: physiological saline in a 1:1 mixture (v/v) FCA/ physiological salineInduction: Topical Application Day 6 - treated group24 hours before application (day 5). A filter paper (2 x 4 cm) with 50% test substance in vaseline was applied to the test area and held in contact by an occlusive dressing for 48 hours. Day 6 – control group24 hours before application (day 5) the test area was cleared of hair. A filter paper (2 x 4 cm) with vaseline only was applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge: Day 20- Exposure period: 24h- Test groups: test substance in vaseline, vaseline- Control group: test substance in vaseline, vaseline- Site: L flank/R flank- Concentrations: 5% substance in vaseline- Evaluation (hr after challenge): 48h and 72hOBSERVATIONS:Clinical signs of intoxication and health condition: dailyMortality/viability: dailyBody weight: 0 and 24th dayThe skin reaction: 24, 48, 72, 96 and 120 hours after intradermal injection48, 72, 96, 120 hours after induction – topical application48 and 72 hours after challenge
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control results:
Reliability of the experimental technique is checked periodically in about six-month interval by the experiment with known sensitiser 2-Mercaptobenzothiazole. The result of last experiment (March 2017): positive skin reaction – 7 animals (total number of animals in exposed group = 10), i.e. 70 %. According to the guideline the minimal number of animals with positive skin reaction should be 30 %. The result is satisfactory.The results of reference study are described in Study No. 413/15/6MK- Ref/03/2017: 2-Mercaptobenzothiazole - Senzibilizace kůže, VUOS-CETA Report No. 17-184.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
5% test substance in vaseline
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
In the course of experiment the animals did not show clinical symptoms of intoxication.
Remarks on result:
positive indication of skin sensitisation
Remarks:
discrete or patchy erythema
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
5 % test substance in vaseline
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
In the course of experiment the animals did not show clinical symptoms of intoxication.
Remarks on result:
positive indication of skin sensitisation
Remarks:
discrete or patchy erythema
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
vaseline only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
In the course of experiment the animals did not show clinical symptoms of intoxication.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
vaseline only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
In the course of experiment the animals did not show clinical symptoms of intoxication.
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Table No. 3: Grading of skin reactions

Exposed

 

 

Control

 

 

Animal No.

Sex

 Start of study

End of study

Animal No.

Sex

Start of study

End of study

11

384.09

520.20

1

422.18

692.90

12

343.95

498.15

2

305.90

470.39

13

331.30

504.91

3

367.05

615.72

14

329.58

519.66

4

434.94

632.96

15

321.99

468.25

5

331.98

584.92

16

349.46

518.55

6

338.86

488.15

17

354.11

488.76

7

375.87

549.48

18

313.27

505.37

8

410.87

544.77

19

352.17

502.38

9

443.82

616.87

20

379.33

536.28

10

375.20

525.78

21

389.73

523.80

22

409.95

536.91

23

324.51

512.13

24

383.07

552.18

25

318.42

507.17

26

327.13

493.86

27

426.25

579.36

28

383.19

577.26

29

325.11

535.23

30

352.03

563.61

♂ – male

♀ – female

0/0 –erythema/oedema

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance, Acid Black 227, caused a positive skin reaction in 7 of 20 animals, i.e. in 35 % of animals, which were exposed to the test substance. Therefore the sensitisation response is positive acc. to criteria in chap. 3.10 of final report.The body weight of animals increased through the study and it was not affected by the treatment. The exposed animals showed no other negative clinical symptoms throughout the experiment.The test substance, Acid Black 227, was shown to be a contact allergen in guinea pigs.
Executive summary:

The test substance, Acid Black 227, was tested for the assessment of skin allergic effects using albino guinea pigs (strain BFA).

The test was performed according to the EU Method B.6, Skin sensitisation. The method is analogous to the OECD Test Guideline No. 406, Skin Sensitisation.

The Magnusson and Kligman maximization procedure was followed. The pilot experiment was implemented on 3 animals. The main test was performed on 20 treated and 10 control animals.

The experiment proceeded in three phases: two induction phases (intradermal injections and topical application) and the challenge phase. Potential skin reactions were evaluated at the end of experiment.

The evaluation of skin reactions in the exposed group carried out at 48 hours after the start of the challenge phase showed discrete erythema in 6 animal in left flank applied with the test substance. Evaluation at 72 hours after the start of the challenge phase of study showed discrete erythema in next 1 animal in left flanks applied with the test substance and in 3 animals the discrete erythema persisted from the previous day.

The body weight of animals increased through the study and it was not affected by the treatment. The exposed animals showed no other negative clinical symptoms throughout the experiment.

The test substance, Acid Black 227, caused a positive skin reaction in 7 of 20 animals, i.e. in 35 % of animals, which were exposed to the test substance. Therefore the sensitisation response is positive. The test substance, Acid Black 227, was shown to be a contact allergen in guinea pigs.